This is a National Phase Application of PCT/EP99/00678 filed Feb. 2, 1999, which in turn, claims priority of EP Application 98105195.6 filed Mar. 23, 1998.
The present invention relates to novel and potentially fluorogenic compounds which become fluorogenic and, hence fluoroscopically detectable upon contact with certain microorganisms or substances produced by such microorganisms, as well as to the use of such compounds for detection and identification of various bacteria.
Conventional assay procedures for identification of bacteria rely on traditional reactions, such as specific characteristics of acid production from particular carbohydrates, esculinase production, pH indicators, and generation of hydrogen sulfide. Such methods tend to be laborious and time consuming and, hence, are costly.
It is known that the enzyme termed xe2x80x9cphosphatidylinositol-specific phospholipase Cxe2x80x9d i.e. 1-phosphatidyl-D-myo-inositol also known as inositolphosphohydrolase, also termed PI-PLC herein for short; enzyme classification EC 3.1.4.10 can be found in culture supernatants of various bacteria including Bacillus thuringiensis as well as some pathogenic bacteria such as Listeria monocytogenes, Listeria ivanovii, Bacillus cereus, Staphylococcus aureus and Clostridium novyi (cf. S. G. Rhee et al, Science 244 (1989) 546 ff).
More recently, Notermans et al. (Applied and Environmental Microbiology 57 (1991), 2666) have reported an assay method based upon analyzing for PI-PLC by overlaying Listeria monocytogenes colonies with a particular substrate, L-xcex1-phosphatidyl-inositol, and examining for turbid halos around the colony indicating the presence of the enzyme. This method is a discontinuous one.
A prior art continuous assay for PI-PLC is based upon the use of 2-naphthyl myoinositol-1-phosphate as a substrate for fluorometric measurement of PI-PLC activity (c.f. M. S. Shashidhar, J. J. Volwerk, J. F. W. Keana, O. H. Griffith; Anal. Biochem. 198 (1991), 10). This substrate has two major disadvantages, however: while 2-naphthol has its maximum fluorescence intensity at pH 10.4, PI-PLC has an optimal pH at about pH 7.4 and is not active above pH 9.0.
Accordingly it is a primary object of the present invention to provide for novel and potentially fluorogenic compounds which avoid the disadvantages of prior art.
The term xe2x80x9cpotentially fluorogenicxe2x80x9d as used herein with reference to the novel compounds according to the invention indicates the capacity of these compounds to become xe2x80x9cfluorogenicxe2x80x9dxe2x80x94i.e. fluoroscopically active and detectable by fluoroscopic methodsxe2x80x94upon interaction with PI-PLC.
Further objects of the invention include improved means for detecting and identifying various pathological bacterias, such as Listeria sp.
The above and further objects and advantages as apparent from the specification will be achieved according to a first embodiment of the invention by potentially fluorogenic compounds of formula (I) 
in which R1,R2,R3,R4 and R5 are independently selected from the group consisting of hydrogen and chromogenic substituents, while X is selected from the group consisting of hydroxyl; OR6 wherein R7 is selected from the group consisting of C1-C4 alkyl; and O-Me+ wherein Me+ is a cation derived from an organic or inorganic base.
While no theoretical limitation is intended, the effectiveness of compounds of formula (I) as substrates for PI-PLC detection is believed to reside in the fact that cleavage of a compound according to the invention by bacterial PI-PLC results mainly in the formation of inositol 1,2-cyclic phosphate and 4-methylumbelliferone which is fluorogenic.
According to a second embodiment, the invention provides for a method of detecting microbial activity in a sample, e.g. in the manner of a screening test, by combining the sample with a compound of formula (I) or a salt thereof and inspecting the sample combined with the formula (I) compound or the salt thereof, preferably as its resulting mixture, by fluoroscopic means.
According to a third embodiment, the invention provides for a method of identifying a bacterial microorganism of interest which is capable of producing a phosphatidyl-inositol-specific phospholipase C enzyme by:
(A) providing a test sample suspected of containing the microorganism of interest;
(B) submitting the test sample to a pre-enrichment step;
(C) combining a portion, at least, of the product obtained in step (B) with a compound of formula (I) to provide a screening sample which exhibits a positive fluoroscopic response when the microorganism of interest or the PI-PLC it produced is present in the test sample;
(D) if the screening sample shows a positive fluoroscopic response in step (B) a portion, at least, of the product obtained in step (B) is transferred to a medium suitable for culturing the microorganism; the medium contains at least one compound capable of producing a colour when exposed to the microorganism;
(E) cultivating the medium with the transferred portion for developing at least one colony exhibiting color; and
(F) recovering a portion, at least, of the coloured colony for final identification.
According to a third embodiment, the invention provides for a substrate for detecting PI-PLC as an indication of bacterial activity; the substrate contains at least one compound of formula (I) in a suitable medium, such as aqueous agar-agar.
According to a fourth object, the invention provides for a kit for detecting PI-PLC as an indication of bacterial activity; the kit includes at least one compound of formula (I).
According to yet a further object, the invention provides for a method of producing a compound of Formula (I) by converting a compound of formula (IIIA) into the compound of formula (IIIB) according to the reaction 
in which xe2x80x9cInsxe2x80x9d represents inositol and G represents a HO-protecting group on each hydroxyl of the inositol except the 1-hydroxy, and wherein X and R1-R5 have the meaning indicated above for formula (I); removing the HO-protecting groups; and optionally forming a salt by reaction with an organic or inorganic base when X represents hydroxyl.
Compounds of formula (I) and (II)xe2x80x94also termed xe2x80x9csubstratesxe2x80x9d herein in view of their interaction with an enzymexe2x80x94may be obtained and used in racemic form and such mixtures can be resolved to obtain the enantiomers. It is to be expected, however, that no substantial advantages will normally be obtained with the enantiomers.
Accordingly, use of racemic mixtures of formula (I) and (II) compounds will be a preferred form of the invention.
Examples of suitable organic and inorganic bases preferred for use according to the invention in its various embodiments including but not restricted to formula (I) when X is hydroxy, or Oxe2x88x92Me+, respectively, are sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide, diethylamine, triethylamine, tetramethylammonium hydroxide, tetraethylammonium hydroxide, cyclohexylamine, pyridine, piperidine, pyrrolidine, morpholine, N-methyl-morpholine, N-ethyl-morpholine and p-toluidine.
A preferred group of compounds of formula (I) are those wherein the chromogenic substituents are: C1-C4 alkyl groups, i.e. methyl, ethyl, propyl and butyl including the isomeric forms, optionally containing an oxygen atom in the alkyl chain; C1-C4 alkoxy; nitro; carboxy, C1-C4 carboxyalkyl; and cyano; optionally, the alkyl groups just mentioned may include one or more halogen atoms, preferably fluorine, chlorine and bromine, as substituents; the trifluoromethyl group is a specific example of a preferred halo-substituted alkyl for use as chromogenic substituent in formula (I).
An even more preferred group of formula (I) compounds includes those wherein R3 is a lower alkyl optionally containing one or more halogen atoms, X is hydroxyl, and R1,R2,R4 and R5 are hydrogen while R3 is a lower alkyl or alkoxy group.
A preferred specific novel compound of formula (I) is 4-methyl umbelliferyl myo-inositol-1-phosphate and the salts thereof with organic or inorganic bases of the type set forth above this compound has the formula: 
A most preferred compound for use as a fluorogenic substrate according to the invention is the N-methyl-morpholine salt of 4-methylumbelliferyl myo-inositol-1-phosphate. It will be referred to as xe2x80x9cMeU-phos-inositolxe2x80x9d herein for brevity. It is a colorless and water-soluble substance having its UV-maximum (in Tris/HCl-buffer at a pH-range of 7 to 9) at 314 nm with an absorption coefficient of ≈10800 1 molxe2x88x921 cmxe2x88x921.
Upon contact with PI-PLC, a potentially fluorogenic compound of formula (I) and (II), respectively, according to the invention becomes a strong fluorophore which can be easily detected, e.g. by a conventional hand held fluorometer, e.g. for operation at a wavelength in the ultraviolet range which is favorable for practical purposes, such as 366 nm. This simple detection approach can be used, according to the invention, either as a screening test and/or as a first step in a more elaborate identification test. In the screening test, lack of fluorescence can be considered a xe2x80x9cnegativexe2x80x9d reaction in the sense that no further testing is required and only a xe2x80x9cpositivexe2x80x9d reaction of the primary samples needs to be verified by additional testing.
More specifically, 4-methylumbelliferone (resulting from cleavage of the formula (II) by PI-PLC) has an absorption maximum of 360 nm at pH values above 8 whereas the corresponding formula (I) compounds show only a negligible absorption at 360 nm.
In other words, 4-methylumbelliferone (4-methyl-7-hydroxycoumarin) is a very good fluorogen. Generally, the intensity of fluorescence of 4-methylumbelliferyl substrates is the same at pH 7 and pH 10 whereas the fluorescence of the product of cleavage increases at pH values in this range. Generally, the enzymatically cleaved substance is preferably determined fluorometrically at a pH near 9.5.
At pH 9.5 typical substrates using 4-Methylumbelliferone as fluorophor show fluorescence emission with maxima around 380 nm (excitation maxima at 325 nm) whereas 4-methylumbelliferone has its fluorescence maximum at 448 nm (excitation maximum 364 nm)
Turning to detection methods according to the invention, the term xe2x80x9cprimary samplexe2x80x9d used herein refers to the material obtained directly from a suspected source that may be of physiological or other origin, such as blood, excrement, or infected foods, water sources, drinks or the like materials capable of harbouring the bacteria of interest. The invention is of particular use for detecting and isolating Listeria, Staphylococcus and Clostridium. An important application of the invention is detection of such human pathogens, as Listeria monocytogenes. 
When using a compound of formula (I) or (II) in a screening or identification test for bacterial activity as evidenced by the presence of PI-PLC, it may be advantageous to provide a pre-enrichment broth in which the primary sample is transferred in order to increase the bacterial activity prior to fluoroscopic analysis. It is preferred for many applications of the inventive method to use a pre-enrichment broth which, in addition, may be selective for the bacteria of interest. The term xe2x80x9cpre-enrichment brothxe2x80x9d which may be but need not be selective is understood by those experienced in the art who are capable of selecting a pre-enrichment broth that is most suitable for the bacteria of interest. Generally, inhibition of growth of other bacteria producing PI-PLC enzyme can be accomplished using various combinations of selective compounds including antibiotics and other inhibitors and the medium can be made specific for any pathogen that contains or produces PI-PLC. More specific examples will be given below.
In both of its aspects as a screening and identification test, respectively, the invention provides for means to indicate bacterial activity of microorganisms having PI-PLC activity, including the nonpathogenic Bacillus thuringiensis as well as the pathogenic Bacillus cereus, Listeria monocytogenes, Listeria ivanovii, Staphylococcus aureus, Clostridium novyi, Trypanosoma brucei. 
As indicated above, a more complete identification step includes the use of a formula (I) compound as a potential fluorogenic agent as well as interaction with a potential chromogenic agent. A colour will be formed in the presence of such a chromogenic agent if bacterial PI-PLC is produced by the bacteria of interest in the culture medium and will localise the colony.
A group of preferred chromogenic agents for producing a colour when exposed to PI-PLC has been disclosed in our and U.S. Pat. Nos. 6,051,391 and 6,068,988, which issued on Apr. 18, 2000 and May 30, 2000, respectively, each based on Provisional Application Serial No. 60/039,479, copending U.S. patent application Ser. No. 60/039 479 and in our international application WO 98/38332, respectively. Generally, such agents have the formula 
wherein R11 is hydrogen or a C1-C4 alkyl and R6, R7, R8, R9 and R9 are selected from hydrogen and chromogenic substituents while R10 is hydrogen or C1-C4 alkyl; again, as in the case of formula (I) compounds, salts of formula (IV) compounds with an organic or inorganic base of the type mentioned above can be used as according to the invention.
In a group of preferred compounds of formula (IV), R6 and R9 are halogen atoms, preferably chlorine and bromine, while R8, R9, R10 are hydrogen; again, the salts of formula (IV) compounds with organic or inorganic bases of the type exemplified above can be used as the potential chromophore, i.e. yielding deeply coloured indigo dyes upon cleavage by PI-PLC, dimerization and subsequent oxidation, especially wherein R10 is hydrogen or methyl and the other substituents hydrogen, fluoro, chloro, bromo, iodo, cyano, nitro, carboxy, substituted or unsubstituted amino, substituted or unsubstituted aminomethyl or sulfonyl.
A most preferred compound of formula (IV) for use as chromogenic substrate according to the present invention are the salts of 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate. The ammonium salt of this compound is termed xe2x80x9cX-phos-inositolxe2x80x9d herein below for brevity.
A preferred test method for identifying Listeria monocytogenes will now be explained in more detail and comprises use of a pre-enrichment broth that can repair or resuscitate injured Listeria monocytogenes cells. After the cells have repaired, the broth can support a rapid growth of Listeria monocytogenes cells. This medium contains inhibitor(s) which will not prevent the repair of injured Listeria monocytogenes cells but can prevent the growth of some Gram negative bacteria that could outcompete Listeria monocytogenes cells for nutrients in the broth.
Then, a selective enrichment broth, that contains the fluorogenic substrate, preferably a 4-methylumbelliferyl myo-inositol-1-phosphate of formula (II) as defined above, is inoculated with a portion, at least, of the pre-enrichment broth. This selective enrichment broth contains inhibitors that prevent growth of other bacteria containing PI-PLC enzyme (other than Listeria ivanovii) and Gram positive bacteria closely related to Listeria monocytogenes. 
Subsequent to incubation, the selective enrichment broth is exposed to a UV fluorometer (long wavelength at 366 nm) and examined for fluorescence. A positive fluorogenic reaction indicates a presumptive positive test requiring further testing and no fluorescence means no Listeria monocytogenes is present in the sample tested (no further testing needed).
After incubation of the selective enrichment and the occurrence of fluorescence, a small portion, e.g. a wire loop transporting a small amount of liquid, is streaked on the selective plating medium containing a potentially chromogenic compound of formula (IV). If Listeria monocytogenes is present, the cells will grow on the plating medium producing a colony which will show the colour developed by of the chromogenic compound when in contact with PI-PLC. Preferably, this plating medium is made selective as well to prevent growth of other bacteria, except Listeria ivanovii, containing the PI-PLC enzyme and Listeria related Gram positive bacteria giving Listeria monocytogenes an optimal environment to produce an isolated colony.
Verification of the chromogenic colony (e.g. for a substrate containing 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphat of formula (IV)) Listeria monocytogenes colony color is turquoise to blue). The colony can be isolated for further testing if required.
The chromogenic substrate works best on a solid surface since the color of the chromogen will be retained within the cell causing the color of the colony to be the color of the chromogen. The chromogen is water insoluble which allows for the color to remain in the colony. Therefore, the chromogenic substrate would not be efficacious in a broth since the color of the chromogen will not cause the color of the broth to change.
Plating media containing X-phos-inositol as chromogenic substrate tend to have very good stability. After 12 weeks of storage at 4xc2x0 C., color and selectivity of the plating medium is the same as that of the freshly prepared medium. The Listeria monocytogenes colonies appear turquoise to blue and convexed, 1.0-2.5 mm in diameter without or with a turquoise to blue halo.
Compounds of formula (I) or their salts can be obtained according to the above reaction scheme by treating, in a first process step, 4-methylumbelliferyl-dichlorophosphate (IIIA) with a reactive inositol compound, e.g. a OH-protected inositol having a free hydroxyl group in 1-position (termed xe2x80x9cG-Ins-OHxe2x80x9d below) so as to obtain an intermediate product, e.g. by stirring the reactants in an organic base, such as pyridine, N-methyl-morpholine or triethylamine, as a reaction medium at ambient temperature during a period of several hours (e.g. 1-10 hours).
G is the protecting group on each hydroxyl of inositol except the 1-hydroxy; typical examples for G include optionally substituted benzyl, optionally substituted C3-6 alkylidene (e.g. isopropylidene, cyclopentylidene or cyclohexylidene); and optionally substituted tetrahydropyranyl.
In the subsequent reaction step, all protecting groups G on the intermediate are removed, e.g. by hydrogenolysis or acidic cleavage depending upon the nature of the OH-protecting groups. Finally, the preferred salt can be obtained by ion exchange.
It will be apparent that such a synthesis method is capable of producing the novel formula (I) compounds efficiently in sufficiently large quantities as are required for application in standard screening procedures.
Based upon storage tests made with the lithium salt and the N-methyl-morpholine salt of 4-Methylumbelliferyl myoinositol-1-phosphate it is to be expected that the novel substrates (I) according to the invention are stable for extended periods of time when stored in a solid state at temperatures below about xe2x88x9215xc2x0 C. and protected from light. Fluorogenic substrates of formula (I) show good stability in a selective enrichment broth medium. When stored in a typical selective enrichment broth at 4xc2x0 C. in the dark, the shelf life of the fluorogenic substrate was 4 weeks.
On the other hand, compounds of formula (I) proved to be only moderately stable at room temperature in conventional buffer solutions (sodium citrate, Tris/HCl) for several hours at pH values ranging from 5 to 7 and are not stable at alkaline pH values. The rate of decomposition increases with the pH. At a pH value of 8.5 the rate of cleavage by PI-PLC is similar to the rate of decomposition (rates≈0.35 xcexcMol 1xe2x88x921 minxe2x88x921 in 1 mM solutions). At pH 9 decomposition proceeds rapidly and at pH values above 10 decomposition is extremely fast.
As a consequence, continuous spectrophotometric monitoring of the cleavage of formula (I) substrates by PI-PLC is not preferred.
On the other hand, a calorimetric assay of PI-PLC using the novel substrates of formula (I) can be conducted effectively at pH values in the range from 6 to 7 where self decomposition in buffer solutions is very slow. Furthermore the enzyme works best at a pH value around 7 and the activity does not vary much between pH 5 and 8.5.
As the absorbance of 4-methylumbelliferone at 360 nm is negligible at a pH of 6, the enzymatically liberated 4-methylumbelliferone is best detected in a discontinuous manner.
Accordingly, enzymic cleavage of the new substrates of formula (I) preferably is conducted at a pH around 6 or 7 and the amount of liberated 4-methylumbelliferone is determined after defined periods of time by raising the pH of a sample to about 9.5, e.g. by addition of a sufficient amount of 1 N sodium hydroxide solution and immediate measurement of the absorbance of the anion of 4-methylumbelliferone with a spectrophotometer.
According to this embodiment of the invention, the novel substrates of formula (I), preferably the N-methylmorpholine salt, are used for a sensitive calorimetric assay of PI-PLC from Bacillus cereus; to this end, the substrate is used in combination with serum albumin, e.g. bovine serum albumin (BSA).
Compared with the prior art substrate 2-NIP mentioned above, MeU-phos-inositol in combination with BSA shows high turnover rates when cleaved by PI-PLC.
It is expected that the novel substrates of formula (I) can be used for a continuous fluorometric assay of PI-PLC: While the intensity of fluorescence of 4-methylumbelliferone has its maximum at pH values above 10, fluorescence intensity is sufficiently high at a pH of 6 or 7 to be utilized for a sensitive assay of PI-PLC.
As a consequence, the fluorogenic substrate (I) tends to be more efficacious in a broth medium than on a solid medium, such as agar, where the fluorescence may leach from the colony into the agar medium causing other nearby colonies to be misread.
Consequently, a positive reaction (secretion of the PI-PLC enzyme by a bacterial species in a broth medium) in the presence of the fluorogenic substrate will cause the broth to show fluorescence indicating that a PI-PLC secreting bacterial species is in the broth at a significant cell density.
This is a presumptive positive reaction for the bacterial species of interest (such as Listeria monocytogenes) which warrant further isolation of the bacterial species in the form of a colony on a solid agar surface using the chromogenic substrate (IV) of the PI-PLC enzyme as explained above.
Use of both a fluorogenic substrates of formula (I) and a chromogenic substrate of formula (IV) in an overall isolation procedure allows for a presumptive positive reaction in the broth (the use of the fluorogenic substrate) followed by isolating the colony on a solid medium using the chromogenic substrate as more of a confirmatory reaction.